Molecular biomarker set for early detection of ovarian cancer

ABSTRACT

Embodiments of the present invention concern methods and compositions related to detection of ovarian cancer, including detection of the stage of ovarian cancer, in some cases. In particular, the invention encompasses use of expression of TFAP2A and in some embodiments CA125 and/or E2F5 to identify ovarian cancer, including detecting mRNA and/or protein levels of the respective gene products. Kits for detection of ovarian cancer are also described.

This application is a continuation of U.S. Non-Provisional patentapplication Ser. No. 13/415,004, filed Mar. 8, 2012, which claimspriority to U.S. Provisional Patent Application Ser. No. 61/450,212,filed Mar. 8, 2011. The entire contents of each of the above-referenceddisclosures are specifically incorporated herein by reference withoutdisclaimer.

TECHNICAL FIELD

The present invention generally concerns at least the fields ofmolecular biology, cell biology, and medicine. In particular aspects,the field concerns detection of cancer, including ovarian cancer.

BACKGROUND OF THE INVENTION

Ovarian cancer is the leading cause of death among gynecologicalmalignancies and represents the fifth leading cause of cancer-relateddeaths in women. The disease is diagnosed at a stage when cancer hasalready metastasized beyond the ovary in approximately 70% of patientsand only 30% of these patients with this advanced-stage ovarian cancersurvive 5 years after initial diagnosis. Early diagnosis greatlyenhances the chances of successful cancer treatment. To this date, veryfew early-detection approaches have shown promise for routine clinicaluse. However, the most commonly used marker of ovarian cancer is CA125,but it is only expressed in 50-60% of patients during early stages ofthe disease.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to methods and compositions thatregard a biomarker set for detection of ovarian cancer in both early andlate stages. In some embodiments, the present invention concernsidentification of the increased risk for an individual to developovarian cancer. In embodiments of the invention, the level of TFAP2A,CA125, and E2F5 (or TFAP2A alone or in other combinations) is assayed inan individual for the detection of ovarian cancer or other cancers. Theindividual may be suspected of having ovarian cancer (as an example),based on, for example, family history, testing, and/or symptoms thatpersist, for example, for two or more weeks (for example, abdominalswelling/bloating; abdominal/pelvic pain or pressure or feeling “full”;gastrointestinal symptoms (such as gas, indigestion, nausea, or changesin bowel movements); vaginal bleeding or discharge; urinary problems(urgency, burning, or spasms); fatigue and/or fever; pain duringintercourse; back pain; and/or difficulty breathing). In someembodiments the individual has no symptoms of cancer, including ovariancancer, yet the individual is subjected to methods of the invention as apart of routine testing.

Individuals with an increased risk of developing ovarian cancer may besubjected to methods of the present invention. In specific cases, anindividual with an increased risk is of a particular age (such as overabout 40, over about 45, over about 50, over about 55, over about 60, orover about 65); has reached menopause; is obese; has no biologicalchildren; has taken fertility drugs or androgens; has had estrogentherapy; has a family history of ovarian and/or breast cancer; has apersonal history of breast cancer; or uses talcum powder on thegenitalia, for example. In some embodiments, the individual has nopersonal or family history of ovarian cancer and is not necessarilyconsidered at risk but undergoes testing at least by methods of theinvention as routine testing in preventative health care (similar toperiodic Pap smear testing for cervical cancer, for example).

In some embodiments, the levels of TFAP2A and optionally one or both ofCA125 and E2F5 are assayed to monitor the effectiveness of an ovariancancer treatment. Exemplary ovarian cancer treatments include surgery,radiation, and/or primary therapy with drugs that contain platinum andtaxane compounds (e.g., cisplatin, carboplatin, paclitaxel); however,other drugs, such as “mustards” (e.g., melphalan) and anthracyclines(e.g., doxorubicin) are also useful for first-line activity in ovariancancer. Other drugs include altretamine, 5-fluorouracil, topotecan,ifosamide, and/or etoposide, for example. The regimen of ovarianchemotherapies are determined by a variety of factors, including thetype and/or stage of ovarian cancer, health status, etc. In someembodiments, the levels of TFAP2A and optionally CA125, and/or E2F5 areutilized for determination of a treatment regimen for ovarian cancer. Insome embodiments of the invention, the methods are employed to determineif an individual should be given a particular cancer therapy, forexample whether an individual would be resistant to a particular cancerdrug.

The present invention allows detection of any type of ovarian cancer, inparticular embodiments. Thus, in certain embodiments the presentinvention may detect ovarian cancers of the three main types of ovariantumors, including epithelial ovarian tumors (the most common), which arederived from the cells on the surface of the ovary; germ cell ovariantumors, which are derived from the egg-producing cells within theovarian body; and sex cord stromal ovarian tumors, which are a type thatoften produces steroid hormones. Epithelial tumors are furthersubdivided into (a) benign, (b) borderline (low-malignant potential[LMP] or atypical proliferative), and (c) invasive carcinoma, and thepresent invention determines any of these types, in specificembodiments. The present invention may be utilized to detect any stageof ovarian cancer, including those identified in the American JointCommittee on Cancer (AJCC) TNM (Tumor size, Lymph Nodes affected,Metastases) system for ovarian cancer: Stage IA, IB, IC, IIA, IIB, IIC,IIIA, IIIB, IIIC, or IV, although in specific embodiments the detectionoccurs for early stage ovarian cancer, such as Stage IA, IB, or IC.

Levels of TFAP2A and optionally CA125 and/or E2F5 may be determined byany suitable means, but in specific embodiments the levels aredetermined for protein or mRNA, or both. The levels may be determinedfrom a sample from the individual, including from a fluid or tissue, forexample. In specific embodiments, the sample is blood or ovarian tissue,such as from a biopsy. The blood may be further fractionated beforeanalysis, in specific embodiments. Samples used in the invention may besubjected to methods of the invention directly from the individual, orthe samples may be stored in a suitable storage means, such as underrefrigeration, and the sample may be transported from the individual toa separate facility for analysis, for example. The person that extractsthe sample may or may not be the person that performs the method(s) ofthe invention, and the facility in which the person that extracts thesample is located may not be the facility in which the method(s) of theinvention is performed.

In some methods of the invention, the method further comprises thestep(s) of obtaining a sample from the individual, isolating nucleicacid (including mRNA) and/or protein from the sample; and analyzing thelevels of TFAP2A and optionally CA125 and/or E2F5 in the sample.

The same sample may be used for processing of each of TFAP2A andoptionally CA125 and/or E2F5, although in other embodiments differentsamples from the same area or type from the same individual are used toanalyze TFAP2A, CA125, and/or E2F5, respectively. The analysis of eachof TFAP2A, CA125, and/or E2F5 may be performed substantiallyconcomitantly or may be performed successively. In some embodiments, asample is analyzed for one or more of TFAP2A and optionally CA125 and/orE2F5, and upon determination of a particular outcome of such analysis,the one or more of TFAP2A, CA125, and/or E2F5, respectively, that werenot originally analyzed are thereafter analyzed.

In one embodiment of the invention, there is a method of evaluating theprobability of the presence of ovarian cancer in a subject, the methodcomprising measuring the amounts of TFAP2A and optionally CA125 and E2F5in a biological sample from the subject; comparing the measured amountsof TFAP2A and optionally CA125 and E2F5 in the biological sample to astandard for TFAP2A and optionally CA125 and E2F5, respectively, whereinthe standard is a level of TFAP2A, CA125, and E2F5, respectively,obtained from a sample of a member of the group consisting of a healthysubject or a subject with normal or benign ovarian tissue, andidentifying an increase in the amount of the respective TFAP2A, CA125,and E2F5 in the biological sample as compared to the standard, whereinthe increase is indicative of the presence of or the probability of thepresence of malignant or pre-malignant cells of ovarian cancer.

In one embodiment of the invention, there is a method of identifyingovarian cancer in an individual or the risk of developing ovarian cancerin an individual, comprising the step of comparing the level of TFAP2Aand optionally CA125 and/or E2F5 from the individual with the expressionlevel of the respective TFAP2A, CA125, and E2F5 from a control. In aspecific embodiment of the invention, when the levels of the respectiveTFAP2A, CA125, and E2F5 are higher in the individual compared to thecontrol, the individual has ovarian cancer or has a risk of developingovarian cancer. In particular aspects of the invention, the proteinlevel of TFAP2A and optionally CA125 and/or E2F5 from the individual isdetermined, such as determined from the blood of the individual. Theprotein level of TFAP2A, CA125, and/or E2F5 may be determined with anantibody, such as a monoclonal antibody, for example.

In some aspects of the invention, the mRNA level of TFAP2A andoptionally CA125 and E2F5 from the individual is determined, such asdetermined from ovarian tissue from the individual. The mRNA level maybe determined by microarray, Northern, or RT-PCR, for example.

In specific embodiments of the invention, the control is from blood ortissue from one or more normal individuals. In particular aspects, themethod detects the stage of ovarian cancer in the individual. In certainembodiments, the stage is stage IA, IC, IIIC, or a combination thereof.In some embodiments, the control is from normal tissue from theindividual being screened.

In some embodiments of the invention, the method further comprises thestep of performing an additional ovarian cancer detection method, suchas one selected from the group consisting of palpitation, ultrasound,magnetic resonance imaging, X-ray, CT scan, blood testing, and biopsy,for example.

In some embodiments, the method further comprises the step ofadministering treatment for ovarian cancer. Exemplary treatments forovarian cancer include surgery, radiation, and/or primary therapy withdrugs that contain platinum and taxane compounds (e.g., cisplatin,carboplatin, paclitaxel); “mustards” (e.g., melphalan), anthracyclines(e.g., doxorubicin), altretamine, 5-fluorouracil, topotecan, ifosamide,and etoposide.

In specific aspects, the method further comprises the step of obtaininga sample from the individual. The sample may be obtained by routinemethods in the art.

In some cases, the method further comprises the step of isolatingTFAP2A, CA125, and E2F5 protein and/or mRNA from the sample. In specificaspects, the method further comprises the steps of obtaining a samplefrom the individual and isolating TFAP2A, CA125, and E2F5 protein and/ormRNA from the sample.

In one embodiment of the invention, there is a kit comprising one ormore detection reagents for TFAP2A, CA125, and E2F5, said reagentshoused in a suitable container, and in some cases the reagent isselected from the group consisting of antibody, microarray,oligonucleotide, polymerase, deoxyribonucleotides, buffer, or acombination thereof. In specific aspects, the method further comprisesan apparatus for obtaining a sample from an individual, such as drawingblood or taking a biopsy from an individual.

In one embodiment, there is a method of identifying ovarian cancer in anindividual or the risk of developing ovarian cancer in an individual,comprising the step of comparing the level of TFAP2A from the individualwith the expression level of TFAP2A from a control. In a specificembodiment, the method further comprises the step of comparing the levelof CA125 or E2F5 or both from the individual with the expression levelof CA125 or E2F5 or both, respectively, from a control.

In a particular embodiment, there is a method of identifying ovariancancer in an individual or the risk of developing ovarian cancer in anindividual, comprising the step of comparing the level of TFAP2A and oneor both of CA125 or E2F5 from the individual with the expression levelof TFAP2A and one or both of CA125 or E2F5, respectively, from acontrol.

In some embodiments, there are methods of identifying ovarian cancer inan individual or the risk of developing ovarian cancer in an individual,comprising the steps of a) obtaining a sample from the individual, b)determining the expression level of TFAP2A from the sample, and c)comparing the expression level of TFAP2A from the individual with theexpression level of TFAP2A from a control, wherein when the level ofTFAP2A is higher in the individual compared to the control, theindividual has ovarian cancer or has a risk of developing ovariancancer.

In some embodiments, there are methods of identifying ovarian cancer inan individual or the risk of developing ovarian cancer in an individual,comprising the steps of a) obtaining a sample from the individual, b)determining the expression level of TFAP2A from the sample, c)determining the expression level of CA125 and/or E2F5 from the sample,d) comparing the expression level of TFAP2A from the individual with theexpression level of TFAP2A from a control, and e) comparing theexpression level of CA125 and/or E2F5 from the individual with theexpression level of CA125 and/or E2F5 from a control, wherein when thelevel of TFAP2A and CA125 or E2F5 is higher in the individual comparedto the control, the individual has ovarian cancer or has a risk ofdeveloping ovarian cancer.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawing.

FIG. 1 shows the comparison of the expression levels of TFAP2A and CA125in randomly chosen ovarian serous adenocarcinoma tissue samplescategorized at stages IA, IC, and IIIC in comparison to normal tissuebased on an ovarian cancer study by Lu et al. (2004).

FIG. 2 shows a bar graph representing the expression pattern of threebiomarkers (TFAP2A, CA125 and E2F5) in normal samples.

FIG. 3 shows the expression pattern of three biomarkers (TFAP2A, CA125and E2F5) in breast cancer samples.

FIG. 4 shows the expression pattern of three biomarkers (TFAP2A, CA125and E2F5) in cervix cancer samples.

FIG. 5 demonstrates the expression pattern of three biomarkers (TFAP2A,CA125 and E2F5) in uterus cancer samples.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. For purposes of the presentinvention, the following terms are defined below.

As used herein, the use of the word “a” or “an” when used in conjunctionwith the term “comprising” in the claims and/or the specification maymean “one,” but it is also consistent with the meaning of “one or more,”“at least one,” and “one or more than one.” Some embodiments of theinvention may consist of or consist essentially of one or more elements,method steps, and/or methods of the invention. It is contemplated thatany method or composition described herein can be implemented withrespect to any other method or composition described herein.

As used herein, the term “biomarker” refers to a marker (expressed gene,including mRNA and/or protein) that allows detection of disease in anindividual when compared to a healthy individual, including detection ofdisease in its early stages. In specific embodiments, the expressionlevel of the biomarker as determined by mRNA and/or protein levels intissue or biological material from an individual to be tested iscompared with respective levels in normal ovarian tissue or biologicalmaterial from the same individual or another healthy individual.

As used herein, the term “control” refers to any entity used incomparison of the biomarkers expression. For example, in the cases whendisease biomarkers are in question, a control could be the expressionpattern of the biomarkers in an individual not affected by the disease;it could be the averaged expression pattern of the biomarkers from agroup of individuals not affected by the disease; it could be theexpression of another gene/protein in the same individual; for onebiomarker it could be the expression of another biomarker in the sameindividual; it could be a threshold on the score produced by amathematical model that uses the expressions of biomarkers and possiblyexpression of other genes/proteins so that scores for disease-affectedindividuals and for individuals not affected by the diseasesignificantly differ (this for example includes all model-basedclassifiers of disease-affected and disease-non-affected cases). Theexpression and the expression pattern could be either absolute orrelative, i.e. determined relative to the expression of some othergene(s)/protein(s). In specific embodiments, the control is derived atleast in part from the level of expression of a reference gene/proteinfrom a single individual without ovarian cancer. One of skill in the artrecognizes that the control expression level may be normalized bystandard means in the art. The normalization may include standardizationto a reference protein (such as a housekeeping gene including GAPDH andRN18S1), for example (see also Tunbridge et al., 2011; Bär et al.,2009). In certain embodiments, the identification of ovarian cancer (oranother respective type of cancer) is achieved when the level ofexpression of a biomarker is above a normalized threshold compared to acontrol. In some cases, the identification of ovarian cancer (or anotherrespective type of cancer) is achieved when the level of expression of abiomarker is below a normalized threshold compared to a control.

In specific embodiments of the invention, the control is from blood ortissue from one or more normal individuals or is from blood or tissuefrom the individual being screened. In specific aspects, the control isderived at least in part from the average level of expression of areference gene/protein in a collection of individuals that do not haveovarian cancer, although the individuals in the collection may or maynot have another type of cancer.

I. General Embodiments of the Invention

The present invention concerns detection of ovarian cancer in anindividual or identifying an individual with an increased risk ofdeveloping ovarian cancer. In some embodiments, the invention offersdiagnostic benefits for patients with ovarian cancer, for example byhelping medical care providers diagnose ovarian cancer in very earlystages that can substantially improve the survival rate in thesepatients. The invention is useful for the health care industry inembodiments where diagnostic kits are routinely used, in specific cases.

Using computational analysis, the inventors have identified TFAP2A,which controls the expression of several ovarian cancer genes in thecell, as a new biomarker for early diagnosis of ovarian cancer. Thecomputation analysis described in this disclosure shows that TFAP2A isuseful as a biomarker for detection of early stages of ovarian cancer,because the data indicate that it is expressed in patients with earlystages of ovarian cancer, but no expression is seen in normalindividuals. For the same set of patients, the analysis shows thatCA125, a known biomarker of ovarian cancer, has detectable expressionlevel in both normal as well as ovarian cancer individuals. Therefore,CA125 does not have the power to differentiate the cancer from normalstates in many cases, whereas the TFAP2A has clearly detectable higherexpression levels in ovarian cancer patients as compared to normal. Acombination of biomarkers TFAP2A, E2F5 and CA125 is a more accuratemeans for diagnosing ovarian cancer.

In certain embodiments of the invention, the technology is based on aunique combination of three biomarkers for use as a diagnostic tool forovarian cancer. All three biomarkers were evaluated individually as wellas in certain combinations for use in diagnosis of cancer. In certainembodiments, the invention concerns identification of a new set of acombination of biomarkers for use in simultaneous diagnosis and earlydetection of ovarian cancer. The biomarker set consists of three geneproducts: TFAP2A, E2F5, and CA125. In specific aspects, the presence ofthese biomarkers in blood, for example, is an indicator of ovariancancer, although other fluids or tissues may be assayed. Differentcombinations of these biomarkers result in different sensitivity andspecificity of ovarian cancer detection. That is, the CA125 is in use asa standard biomarker for detection of ovarian cancer, but it does nothave good accuracy of ovarian cancer detection (Sasaroli et al., 2009).Another biomarker, E2F5, has been recently discovered (Kothandaraman etal., 2010) where it has been shown that in combination with CA125 therewas significant improvement of the accuracy of ovarian cancer detectionand ability to detect ovarian cancer in early stages. The thirdbiomarker for use in combination with CA125 and E2F5 is TFAP2A thatshows expression in early ovarian cancer stages but not in normalindividuals.

In some embodiments of the invention, there is a single biomarker usedas a diagnostic tool for cancer, including ovarian cancer. In specificaspects, the presence of this biomarker in blood, for example, is anindicator of ovarian cancer, although other fluids or tissues may beassayed.

Thus, in embodiments of the invention, presence of TFAP2A, E2F5, andCA125 in different combinations in human blood samples improves theaccuracy of diagnosis of ovarian cancer in clinical settings andenhances the capability to detect ovarian cancer, for example in earlystages.

In some embodiments, the mere presence or absence of a marker, withoutquantifying the amount of marker, is useful and can be correlated with adiagnosis of ovarian cancer or increased risk of developing ovariancancer. For example, TFAP2A, E2F5, and CA125 can be more frequentlydetected in human ovarian cancer patients than in normal subjects. Thus,a detected presence of these markers in a subject being tested indicatesthat the subject has ovarian cancer or has a higher probability ofhaving ovarian cancer.

In other embodiments, the measurement of markers can involve quantifyingthe markers to correlate the detection of markers with a probablediagnosis of ovarian cancer. Thus, if the amount of the markers detectedin a subject being tested is different compared to a control amount(i.e., higher or lower than the control, depending on the marker), thenthe subject being tested has a higher probability of having ovariancancer.

The correlation may take into account the amount of the marker ormarkers in the sample compared to a control amount of the marker ormarkers (up or down regulation of the marker or markers) (e.g., innormal subjects in whom human cancer is undetectable). A control can be,e.g., the average or median amount of marker present in comparablesamples of normal subjects in whom human cancer is undetectable. Thecontrol amount is measured under the same or substantially similarexperimental conditions as in measuring the test amount. The correlationmay take into account the presence or absence of the markers in a testsample and the frequency of detection of the same markers in a control.The correlation may take into account both of such factors to facilitatedetermination of ovarian cancer status.

In certain embodiments of the methods of qualifying ovarian cancerstatus, the methods further comprise managing subject treatment based onthe status. As aforesaid, such management describes the actions of thephysician or clinician subsequent to determining ovarian cancer status.For example, if the result of the methods of the present invention isinconclusive or there is reason that confirmation of status isnecessary, the physician may order more tests. Alternatively, if thestatus indicates that surgery is appropriate, the physician may schedulethe patient for surgery. In other instances, the patient may receivechemotherapy or radiation treatments, either in lieu of, or in additionto, surgery. Likewise, if the result is negative, no further action maybe warranted. Furthermore, if the results show that treatment has beensuccessful, no further management may be necessary.

The invention also provides for such methods where the biomarkers (orspecific combination of biomarkers) are measured again after subjectmanagement. In these cases, the methods are used to monitor the statusof the cancer, e.g., response to cancer treatment, remission of thedisease or progression of the disease. Because of the ease of use of themethods and the lack of invasiveness at least in certain embodiments ofthe methods, the methods can be repeated after each treatment thepatient receives. This allows the physician to follow the effectivenessof the course of treatment. If the results show that the treatment isnot effective, the course of treatment can be altered accordingly. Thisenables the physician to be flexible in the treatment options.

The methods of the present invention have other applications as well.For example, the markers can be used to screen for compounds thatmodulate the expression of the markers in vitro or in vivo, whichcompounds in turn may be useful in treating or preventing ovarian cancerin patients. In another example, the markers can be used to monitor theresponse to treatments for ovarian cancer. In yet another example, themarkers can be used in heredity studies to determine if the subject isat risk for developing ovarian cancer. For instance, certain markers maybe genetically linked. This can be determined by, e.g., analyzingsamples from a population of ovarian cancer patients whose families havea history of ovarian cancer. The results can then be compared with dataobtained from, e.g., ovarian cancer patients whose families do not havea history of ovarian cancer. The markers that are genetically linked maybe used as a tool to determine if a subject whose family has a historyof ovarian cancer is pre-disposed to having ovarian cancer.

II. Gene Products Used in Embodiments of the Invention

The present invention employs methods and reagents related to assayingfor expression levels of one or more particular genes, including TFAP2Aand, in embodiments of the invention, also of CA125 and E2F5. Theexpression level may be determined by measuring levels of mRNA and/orprotein, in specific embodiments.

TFAP2A is also referred to as transcription factor AP-2 alpha(activating enhancer binding protein 2 alpha); RP1-290I10.1; AP-2;AP-2alpha; AP2TF; BOFS; FLJ51761; TFAP2; AP-2 transcription factor;AP2-alpha; OTTHUMP00000214240; OTTHUMP00000214243; activatingenhancer-binding protein 2-alpha; and activator protein 2; for example.Exemplary GenBank® TFAP2A mRNA sequences are provided in NM_001032280.2;NM_001042425.1; and NM_003220.2 (SEQ ID NO:1) (the three of whichcorrespond to isoforms b, c, and a, respectively), all of whichsequences are incorporated by reference herein. Exemplary GenBank®TFAP2A protein sequences are provided in NP_001027451.1; NP_001035890.1;and NP_003211.1 (SEQ ID NO:2) (the three of which correspond to isoformsb, c, and a, respectively), all of which sequences are incorporated byreference herein.

E2F5 is also referred to as E2F transcription factor 5, E2F-5, andp130-binding. Exemplary GenBank® E2F5 mRNA sequences are provided inNM_001083588.1; NM_001083589.1; and NM_001951.3 (SEQ ID NO:3) (the threeof which correspond to isoforms 2, 3, and 1, respectively), all of whichsequences are incorporated by reference herein. Exemplary GenBank®E2F5protein sequences are provided in NP_001077057.1; NP_001077058.1; andNP_001942.2 (SEQ ID NO:4) (the three of which correspond to isoforms 2,3, and 1, respectively), all of which sequences are incorporated byreference herein.

CA125 is also referred to as cancer antigen 125; carbohydrate antigen125; MUC16; mucin 16, cell surface associated; FLJ14303; CA-125; CA125ovarian cancer antigen; MUC-16; mucin-16; ovarian cancer-related tumormarker CA125; and ovarian carcinoma antigen CA125. Exemplary GenBank®CA125 mRNA sequence is provided in NM_024690.2 (SEQ ID NO:5), which isincorporated by reference herein. Exemplary GenBank® CA125 proteinsequence is provided in NP_078966.2 (SEQ ID NO:6), which is incorporatedby reference herein.

Other Markers

In some embodiments of the invention, additional markers to TFAP2A,E2F5, and CA125 are utilized in the invention. In exemplary embodiments,ovarian cancer markers described in U.S. Pat. No. 7,605,003, which isincorporated herein by reference, are employed in the invention.Exemplary markers include CA15-3, CA19-9, CA72-4, CA 195, TATI, CEA,PLAP, Sialyl TN, galactosyltransferase, M-CSF, CSF-1, LPA, p110EGFR,tissue kallikreins, prostasin, HE4, CKB, LASA, HER-2/neu, urinarygonadotropin peptide, Dianon NB 70/K, TPA, osteopontin and haptoglobin,and protein variants (e.g., cleavage forms, isoforms) of the markers; inspecific embodiments, the levels of these markers are different inindividuals with cancer compared to normal individuals. For example, CA19-9, CA 72.4, CA 195, TATI, inhibin and PLAP, and others, are known tobe elevated in the blood of women with ovarian cancer.

Other markers that may be used in the methods of the invention includethose described in U.S. Pat. No. 7,745,149, which is incorporated byreference herein, and encompasses galectin-1, cathepsin B, MHC class Iantigen, heat shock protein (HSP) 27, ubiquitin carboxy-termal esteraseL1, plasma retinol-binding protein (PRBP), transthyretin, SH3 bindingglutamate-rich protein, tubulin-specific chaperone A, RNA bindingprotein regulatory subunit, γ-actin, tropomyosin andcalcium/calmodulin-stimulated cyclic nucleotide phosphatase.

Additional markers that may be used in the methods of the inventioninclude hK10 and/or hK6, described in U.S. Pat. No. 7,741,019, which isincorporated by reference herein.

III. Test Samples

A. Subject Types

Samples are collected from subjects, e.g., women, who want to establishovarian cancer status. The subjects may be women who have beendetermined to have a high risk of ovarian cancer, for example based ontheir family history. Other patients include women who have ovariancancer and the test is being used to determine the effectiveness oftherapy or treatment they are receiving. Also, patients could includehealthy women who are having a test as part of a routine examination, orto establish baseline levels of the biomarkers. Samples may be collectedfrom women who had been diagnosed with ovarian cancer and receivedtreatment to eliminate the cancer, or perhaps are in remission.

B. Types of Sample and Preparation of the Sample

The markers can be measured in different types of biological samples.The sample is preferably a biological fluid sample. Examples of abiological fluid sample useful in this invention include blood, bloodserum, plasma, vaginal secretions, urine, tears, saliva, etc. Blood is apreferred sample source for certain embodiments of the invention becauseof its non-invasiveness. In alternative embodiments, the sample is frombiopsy.

If desired, the sample can be prepared to enhance detectability of themarkers. For example, to increase the detectability of markers, a bloodserum sample from the subject can be preferably fractionated by, e.g.,Cibacron blue agarose chromatography and single stranded DNA affinitychromatography, anion exchange chromatography, affinity chromatography(e.g., with antibodies) and the like. The method of fractionationdepends on the type of detection method used. Any method that enrichesfor the protein of interest can be used. Sample preparations, such aspre-fractionation protocols, are optional and may not be necessary toenhance detectability of markers depending on the methods of detectionused, in some embodiments. For example, sample preparation may beunnecessary if antibodies that specifically bind markers are used todetect the presence of markers in a sample.

Typically, sample preparation involves fractionation of the sample andcollection of fractions determined to contain the biomarkers. Methods ofpre-fractionation include, for example, size exclusion chromatography,ion exchange chromatography, heparin chromatography, affinitychromatography, sequential extraction, gel electrophoresis and liquidchromatography. The analytes also may be modified prior to detection.These methods are useful to simplify the sample for further analysis.For example, it can be useful to remove high abundance proteins, such asalbumin, from blood before analysis. Examples of methods offractionation are described in PCT/US03/00531 (incorporated herein inits entirety).

In some cases, the sample is pre-fractionated by anion exchangechromatography. Anion exchange chromatography allows pre-fractionationof the proteins in a sample roughly according to their chargecharacteristics. For example, a Q anion-exchange resin can be used(e.g., Q HyperD F, Biosepra), and a sample can be sequentially elutedwith eluants having different pH's. Anion exchange chromatography allowsseparation of biomolecules in a sample that are more negatively chargedfrom other types of biomolecules. Proteins that are eluted with aneluant having a high pH is likely to be weakly negatively charged, and afraction that is eluted with an eluant having a low pH is likely to bestrongly negatively charged. Thus, in addition to reducing complexity ofa sample, anion exchange chromatography separates proteins according totheir binding characteristics.

In certain embodiments, the serum samples are fractionated via anionexchange chromatography. Signal suppression of lower abundance proteinsby high abundance proteins presents a significant challenge to SELDImass spectrometry. Fractionation of a sample reduces the complexity ofthe constituents of each fraction. This method can also be used toattempt to isolate high abundance proteins into a fraction, and therebyreduce its signal suppression effect on lower abundance proteins. Anionexchange fractionation separates proteins by their isoelectric point(pI). Proteins are comprised of amino acids, which are ambivalent-theircharge changes based on the pH of the environment to which they areexposed. A protein's pI is the pH at which the protein has no netcharge. A protein assumes a neutral charge when the pH of theenvironment is equivalent to pI of the protein. When the pH rises abovethe pI of the protein, the protein assumes a net negative charge.Similarly, when the pH of the environment falls below the pH of theprotein, the protein has a net positive charge. The serum samples werefractionated according to the protocol set forth in the Examples belowto obtain the markers described herein.

After capture on anion exchange, proteins were eluted in a series ofstep washes at pH 9, pH 7, pH 5, pH 4 and pH 3. A panel of threepotential biomarkers was discovered by UMSA analysis of profiling dataof three fractions (pH 9/flow through, pH 4, and organic solvent). Twoof the peaks were from fraction pH 4 at m/z of 12828 and 28043, bothdown-regulated in the cancer group, and the third was from fraction pH9/flow through at m/z of 3272, up-regulated in the cancer group. Allbound to the immobilized metal affinity chromatography array chargedwith copper ions (IMAC3-Cu) (spectra in FIG. 1).

Biomolecules in a sample can also be separated by high-resolutionelectrophoresis, e.g., one or two-dimensional gel electrophoresis. Afraction containing a marker can be isolated and further analyzed by gasphase ion spectrometry. Preferably, two-dimensional gel electrophoresisis used to generate two-dimensional array of spots of biomolecules,including one or more markers. See, e.g., Jungblut and Thiede, MassSpecir. Rev. 16:145-162 (1997).

The two-dimensional gel electrophoresis can be performed using methodsknown in the art. See, e.g., Deutscher ed., Methods In Enzymology vol.182. Typically, biomolecules in a sample are separated by, e.g.,isoelectric focusing, during which biomolecules in a sample areseparated in a pH gradient until they reach a spot where their netcharge is zero (i.e., isoelectric point). This first separation stepresults in one-dimensional array of biomolecules. The biomolecules inone-dimensional array is further separated using a technique generallydistinct from that used in the first separation step. For example, inthe second dimension, biomolecules separated by isoelectric focusing arefurther separated using a polyacrylamide gel, such as polyacrylamide gelelectrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE).SDS-PAGE gel allows further separation based on molecular mass ofbiomolecules. Typically, two-dimensional gel electrophoresis canseparate chemically different biomolecules in the molecular mass rangefrom 1000-200,000 Da within complex mixtures. The pI range of these gelsis about 3-10 (wide range gels).

Biomolecules in the two-dimensional array can be detected using anysuitable methods known in the art. For example, biomolecules in a gelcan be labeled or stained (e.g., Coomassie Blue or silver staining). Ifgel electrophoresis generates spots that correspond to the molecularweight of one or more markers of the invention, the spot can be furtheranalyzed by gas phase ion spectrometry. For example, spots can beexcised from the gel and analyzed by gas phase ion spectrometry.Alternatively, the gel containing biomolecules can be transferred to aninert membrane by applying an electric field. Then a spot on themembrane that approximately corresponds to the molecular weight of amarker can be analyzed by gas phase ion spectrometry. In gas phase ionspectrometry, the spots can be analyzed using any suitable techniques,such as MALDI or SELDI (e.g., using ProteinChip® array) as describedherein.

Prior to gas phase ion spectrometry analysis, it may be desirable tocleave biomolecules in the spot into smaller fragments using cleavingreagents, such as proteases (e.g., trypsin). The digestion ofbiomolecules into small fragments provides a mass fingerprint of thebiomolecules in the spot, which can be used to determine the identity ofmarkers if desired.

High performance liquid chromatography (HPLC) can also be used toseparate a mixture of biomolecules in a sample based on their differentphysical properties, such as polarity, charge and size. HPLC instrumentstypically consist of a reservoir of mobile phase, a pump, an injector, aseparation column, and a detector. Biomolecules in a sample areseparated by injecting an aliquot of the sample onto the column.Different biomolecules in the mixture pass through the column atdifferent rates due to differences in their partitioning behaviorbetween the mobile liquid phase and the stationary phase. A fractionthat corresponds to the molecular weight and/or physical properties ofone or more markers can be collected. The fraction can then be analyzedby gas phase ion spectrometry to detect markers. For example, the spotscan be analyzed using either MALDI or SELDI (e.g., using ProteinChip®array) as described herein.

Optionally, a marker can be modified before analysis to improve itsresolution or to determine its identity. For example, the markers may besubject to proteolytic digestion before analysis. Any protease can beused. Proteases, such as trypsin, that are likely to cleave the markersinto a discrete number of fragments are particularly useful. Thefragments that result from digestion function as a fingerprint for themarkers, thereby enabling their detection indirectly. This isparticularly useful where there are markers with similar molecularmasses that might be confused for the marker in question. Also,proteolytic fragmentation is useful for high molecular weight markersbecause smaller markers are more easily resolved by mass spectrometry.In another example, biomolecules can be modified to improve detectionresolution. For instance, neuraminidase can be used to remove terminalsialic acid residues from glycoproteins to improve binding to an anionicadsorbent (e.g., cationic exchange ProteinChip® arrays) and to improvedetection resolution. In another example, the markers can be modified bythe attachment of a tag of particular molecular weight that specificallybind to molecular markers, further distinguishing them. Optionally,after detecting such modified markers, the identity of the markers canbe further determined by matching the physical and chemicalcharacteristics of the modified markers in a protein database (e.g.,SwissProt).

C. Capture of Markers

Biomarkers may be captured with capture reagents immobilized to a solidsupport, such as any biochip described herein, a multiwell microtiterplate or a resin. In particular, the biomarkers of this invention arepreferably captured on SELDI protein biochips. Capture can be on achromatographic surface or a biospecific surface. Any of the SELDIprotein biochips comprising reactive surfaces can be used to capture anddetect the biomarkers of this invention. However, the biomarkers of thisinvention bind well to immobilized metal chelates. The IMAC-3 and IMAC30 biochips, which nitriloacetic acid functionalities that adsorbtransition metal ions, such as Cu++ and Ni++, by chelation, are thepreferred SELDI biochips for capturing the biomarkers of this invention.Any of the SELDI protein biochips comprising reactive surfaces can beused to capture and detect the biomarkers of this invention. Thesebiochips can be derivatized with the antibodies that specificallycapture the biomarkers, or they can be derivatized with capturereagents, such as protein A or protein G that bind immunoglobulins. Thenthe biomarkers can be captured in solution using specific antibodies andthe captured markers isolated on chip through the capture reagent.

In general, a sample containing the biomarkers, such as blood or serum,is placed on the active surface of a biochip for a sufficient time toallow binding. Then, unbound molecules are washed from the surface usinga suitable eluant, such as phosphate buffered saline. In general, themore stringent the eluant, the more tightly the proteins must be boundto be retained after the wash. The retained protein biomarkers now canbe detected by appropriate means.

D. Detection and Measurement of Markers

Once captured on a substrate, e.g., biochip or antibody, any suitablemethod can be used to measure a marker or markers in a sample. Forexample, markers can be detected and/or measured by a variety ofdetection methods including for example, gas phase ion spectrometrymethods, optical methods, electrochemical methods, atomic forcemicroscopy and radio frequency methods. Using these methods, one or moremarkers can be detected.

1. SELDI

One preferred method of detection and/or measurement of the biomarkersuses mass spectrometry and, in particular, “Surface-enhanced laserdesorption/ionization” or “SELDI”. SELDI refers to a method ofdesorption/ionization gas phase ion spectrometry (e.g., massspectrometry) in which the analyte is captured on the surface of a SELDIprobe that engages the probe interface. In “SELDI MS,” the gas phase ionspectrometer is a mass spectrometer. SELDI technology is described inmore detail above. ApoA1, transthyretin .DELTA.N10 and IAIH4 fragmentare detected as peaks at m/z of 28043, m/z of about 12870.9, and m/z of3272, respectively.

2. Immunoassay

In another embodiment, an immunoassay can be used to detect and analyzemarkers in a sample. This method comprises: (a) providing an antibodythat specifically binds to a marker; (b) contacting a sample with theantibody; and (c) detecting the presence of a complex of the antibodybound to the marker in the sample.

An immunoassay is an assay that uses an antibody to specifically bind anantigen (e.g., a marker). The immunoassay is characterized by the use ofspecific binding properties of a particular antibody to isolate, target,and/or quantify the antigen. The phrase “specifically (or selectively)binds” to an antibody or “specifically (or selectively) immunoreactivewith,” when referring to a protein or peptide, refers to a bindingreaction that is determinative of the presence of the protein in aheterogeneous population of proteins and other biologics. Thus, underdesignated immunoassay conditions, the specified antibodies bind to aparticular protein at least two times the background and do notsubstantially bind in a significant amount to other proteins present inthe sample. Specific binding to an antibody under such conditions mayrequire an antibody that is selected for its specificity for aparticular protein. For example, polyclonal antibodies raised to amarker from specific species such as rat, mouse, or human can beselected to obtain only those polyclonal antibodies that arespecifically immunoreactive with that marker and not with otherproteins, except for polymorphic variants and alleles of the marker.This selection may be achieved by subtracting out antibodies thatcross-react with the marker molecules from other species.

Using the purified markers or their nucleic acid sequences, antibodiesthat specifically bind to a marker can be prepared using any suitablemethods known in the art. See, e.g., Coligan, Current Protocols inImmunology (1991); Harlow & Lane, Antibodies: A Laboratory Manual(1988); Goding, Monoclonal Antibodies: Principles and Practice (2d ed.1986); and Kohler & Milstein, Nature 256:495-497 (1975). Such techniquesinclude, but are not limited to, antibody preparation by selection ofantibodies from libraries of recombinant antibodies in phage or similarvectors, as well as preparation of polyclonal and monoclonal antibodiesby immunizing rabbits or mice (see, e.g., Huse et al., Science246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989)). Typicallya specific or selective reaction will be at least twice backgroundsignal or noise and more typically more than 10 to 100 times background.

Generally, a sample obtained from a subject can be contacted with theantibody that specifically binds the marker. Optionally, the antibodycan be fixed to a solid support to facilitate washing and subsequentisolation of the complex, prior to contacting the antibody with asample. Examples of solid supports include glass or plastic in the formof, e.g., a microtiter plate, a stick, a bead, or a microbead.Antibodies can also be attached to a probe substrate or ProteinChip®array described above. The sample is preferably a biological fluidsample taken from a subject. Examples of biological fluid samplesinclude blood, serum, plasma, nipple aspirate, urine, tears, saliva etc.In a preferred embodiment, the biological fluid comprises blood serum.The sample can be diluted with a suitable eluant before contacting thesample to the antibody.

After incubating the sample with antibodies, the mixture is washed andthe antibody-marker complex formed can be detected. This can beaccomplished by incubating the washed mixture with a detection reagent.This detection reagent may be, e.g., a second antibody which is labeledwith a detectable label. Exemplary detectable labels include magneticbeads (e.g., DYNABEADS™), fluorescent dyes, radiolabels, enzymes (e.g.,horse radish peroxide, alkaline phosphatase and others commonly used inan ELISA), and colorimetric labels such as colloidal gold or coloredglass or plastic beads. Alternatively, the marker in the sample can bedetected using an indirect assay, wherein, for example, a second,labeled antibody is used to detect bound marker-specific antibody,and/or in a competition or inhibition assay wherein, for example, amonoclonal antibody which binds to a distinct epitope of the marker isincubated simultaneously with the mixture.

Methods for measuring the amount of, or presence of, antibody-markercomplex include, for example, detection of fluorescence, luminescence,chemiluminescence, absorbance, reflectance, transmittance, birefringenceor refractive index (e.g., surface plasmon resonance, ellipsometry, aresonant mirror method, a grating coupler waveguide method orinterferometry). Optical methods include microscopy (both confocal andnon-confocal), imaging methods and non-imaging methods. Electrochemicalmethods include voltametry and amperometry methods. Radio frequencymethods include multipolar resonance spectroscopy. Methods forperforming these assays are readily known in the art. Useful assaysinclude, for example, an enzyme immune assay (EIA) such as enzyme-linkedimmunosorbent assay (ELISA), a radioimmune assay (RIA), a Western blotassay, or a slot blot assay. These methods are also described in, e.g.,Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai,ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed.1991); and Harlow & Lane, supra.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,marker, volume of solution, concentrations and the like. Usually theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

Immunoassays can be used to determine presence or absence of a marker ina sample as well as the quantity of a marker in a sample. The amount ofan antibody-marker complex can be determined by comparing to a standard.A standard can be, e.g., a known compound or another protein known to bepresent in a sample. As noted above, the test amount of marker need notbe measured in absolute units, as long as the unit of measurement can becompared to a control.

The methods for detecting these markers in a sample have manyapplications. For example, one or more markers can be measured to aidhuman cancer diagnosis or prognosis. In another example, the methods fordetection of the markers can be used to monitor responses in a subjectto cancer treatment. In another example, the methods for detectingmarkers can be used to assay for and to identify compounds that modulateexpression of these markers in vivo or in vitro. In a preferred example,the biomarkers are used to differentiate between the different stages oftumor progression, thus aiding in determining appropriate treatment andextent of metastasis of the tumor.

IV. Combination Methods for Detection of Ovarian Cancer

In embodiments of the invention, the results of the inventive detectionmethods are employed with other detection methods or information toprovide a diagnosis for an individual. Exemplary ovarian cancerdetection methods include analysis of blood, urine or a biopsy of asuspicious area. Examples of blood and urine tests used in combinationwith methods of the invention include, for example, complete blood count(CBC); urine cytology; blood protein testing (for example, to detectcertain abnormal immune system proteins (immunoglobulins); and tumormarker tests. In some embodiments of the invention, an individual with apelvic mass is subjected to one or more methods of the invention.

Although in some embodiments of the invention the gene product levelsare used to monitor patients with a known cancer, for example todetermine the stage of cancer or to monitor effectiveness of a cancertherapy, in other embodiments the methods are employed as one of severaltests in the workup of an individual suspected of having a tumor.

Thus, in individuals who are known to have a malignancy, such as ovariancancer, the TFAP2A, E2F5, and CA 125 levels can be monitoredperiodically, for example. A changed level generally may indicate thattherapy, including chemotherapy, has been effective, a level changed inthe opposite direction may indicate tumor recurrence, while a stagnantlevel may indicate lack of effectiveness.

V. Nucleic Acid Detection

In some embodiments of the invention, the expression of TFAP2A, E2F5,and CA125 nucleic acid sequences disclosed herein is determined forovarian cancer detection or propensity for developing ovarian cancer.

A. Hybridization

In some embodiments, hybridization of respective probes to TFAP2A, E2F5,and CA125 mRNAs are employed in the invention. The use of a probe orprimer of between 13 and 100 nucleotides, preferably between 17 and 100nucleotides in length, or in some aspects of the invention up to 1-2kilobases or more in length, allows the formation of a duplex moleculethat is both stable and selective. Molecules having complementarysequences over contiguous stretches greater than 20 bases in length aregenerally preferred, to increase stability and/or selectivity of thehybrid molecules obtained. One will generally prefer to design nucleicacid molecules for hybridization having one or more complementarysequences of 20 to 30 nucleotides, or even longer where desired. Suchfragments may be readily prepared, for example, by directly synthesizingthe fragment by chemical means or by introducing selected sequences intorecombinant vectors for recombinant production.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNAs and/or RNAs or to provide primers for amplification ofDNA or RNA from samples. Depending on the application envisioned, onewould desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of the probe or primers for the targetsequence.

For applications requiring high selectivity, one will typically desireto employ relatively high stringency conditions to form the hybrids. Forexample, relatively low salt and/or high temperature conditions, such asprovided by about 0.02 M to about 0.10 M NaCl at temperatures of about50° C. to about 70° C. Such high stringency conditions tolerate little,if any, mismatch between the probe or primers and the template or targetstrand and would be particularly suitable for isolating specific genesor for detecting specific mRNA transcripts. It is generally appreciatedthat conditions can be rendered more stringent by the addition ofincreasing amounts of formamide.

For certain applications, for example, site-directed mutagenesis, it isappreciated that lower stringency conditions are preferred. Under theseconditions, hybridization may occur even though the sequences of thehybridizing strands are not perfectly complementary, but are mismatchedat one or more positions. Conditions may be rendered less stringent byincreasing salt concentration and/or decreasing temperature. Forexample, a medium stringency condition could be provided by about 0.1 to0.25 M NaCl at temperatures of about 37° C. to about 55° C., while a lowstringency condition could be provided by about 0.15 M to about 0.9 Msalt, at temperatures ranging from about 20° C. to about 55° C.Hybridization conditions can be readily manipulated depending on thedesired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 1.0 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, attemperatures ranging from approximately 40° C. to about 72° C.

In certain embodiments, it will be advantageous to employ nucleic acidsof defined sequences of the present invention in combination with anappropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including fluorescent, radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of being detected. In preferredembodiments, one may desire to employ a fluorescent label or an enzymetag such as urease, alkaline phosphatase or peroxidase, instead ofradioactive or other environmentally undesirable reagents. In the caseof enzyme tags, colorimetric indicator substrates are known that can beemployed to provide a detection means that is visibly orspectrophotometrically detectable, to identify specific hybridizationwith complementary nucleic acid containing samples.

In general, it is envisioned that the probes or primers described hereinwill be useful as reagents in solution hybridization, as in PCR®, fordetection of expression of corresponding genes, as well as inembodiments employing a solid phase. In embodiments involving a solidphase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to hybridization with selected probes under desiredconditions. The conditions selected will depend on the particularcircumstances (depending, for example, on the G+C content, type oftarget nucleic acid, source of nucleic acid, size of hybridizationprobe, etc.). Optimization of hybridization conditions for theparticular application of interest is well known to those of skill inthe art. After washing of the hybridized molecules to removenon-specifically bound probe molecules, hybridization is detected,and/or quantified, by determining the amount of bound label.Representative solid phase hybridization methods are disclosed in U.S.Pat. Nos. 5,843,663, 5,900,481 and 5,919,626. Other methods ofhybridization that may be used in the practice of the present inventionare disclosed in U.S. Pat. Nos. 5,849,481, 5,849,486 and 5,851,772. Therelevant portions of these and other references identified in thissection of the Specification are incorporated herein by reference.

B. Amplification of Nucleic Acids

Nucleic acids used as a template for amplification may be isolated fromcells, tissues or other samples according to standard methodologies(Sambrook et al., 1989). In certain embodiments, analysis is performedon whole cell or tissue homogenates or biological fluid samples withoutsubstantial purification of the template nucleic acid. The nucleic acidmay be genomic DNA or fractionated or whole cell RNA. Where RNA is used,it may be desired to first convert the RNA to a complementary DNA.

The term “primer,” as used herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom ten to twenty and/or thirty base pairs in length, but longersequences can be employed. Primers may be provided in double-strandedand/or single-stranded form, although the single-stranded form ispreferred.

Pairs of primers designed to selectively hybridize to nucleic acidscorresponding to TFAP2A, E2F5, and CA125 are contacted with the templatenucleic acid under conditions that permit selective hybridization.Depending upon the desired application, high stringency hybridizationconditions may be selected that will only allow hybridization tosequences that are completely complementary to the primers. In otherembodiments, hybridization may occur under reduced stringency to allowfor amplification of nucleic acids contain one or more mismatches withthe primer sequences. Once hybridized, the template-primer complex iscontacted with one or more enzymes that facilitate template-dependentnucleic acid synthesis. Multiple rounds of amplification, also referredto as “cycles,” are conducted until a sufficient amount of amplificationproduct is produced.

The amplification product may be detected or quantified. In certainapplications, the detection may be performed by visual means.Alternatively, the detection may involve indirect identification of theproduct via chemiluminescence, radioactive scintigraphy of incorporatedradiolabel or fluorescent label or even via a system using electricaland/or thermal impulse signals (Affymax technology; Bellus, 1994).

A number of template dependent processes are available to amplify theoligonucleotide sequences present in a given template sample. One of thebest known amplification methods is the polymerase chain reaction(referred to as PCR™) which is described in detail in U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159, and in Innis et al., 1988, each ofwhich is incorporated herein by reference in their entirety.

A reverse transcriptase PCR™ amplification procedure may be performed toquantify the amount of mRNA amplified. Methods of reverse transcribingRNA into cDNA are well known (see Sambrook et al., 1989). Alternativemethods for reverse transcription utilize thermostable DNA polymerases.These methods are described in WO 90/07641. Polymerase chain reactionmethodologies are well known in the art. Representative methods ofRT-PCR are described in U.S. Pat. No. 5,882,864.

Another method for amplification is ligase chain reaction (“LCR”),disclosed in European Application No. 320 308, incorporated herein byreference in its entirety. U.S. Pat. No. 4,883,750 describes a methodsimilar to LCR for binding probe pairs to a target sequence. A methodbased on PCR™ and oligonucleotide ligase assy (OLA), disclosed in U.S.Pat. No. 5,912,148, may also be used.

Alternative methods for amplification of target nucleic acid sequencesthat may be used in the practice of the present invention are disclosedin U.S. Pat. Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497,5,849,547, 5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905,5,928,906, 5,932,451, 5,935,825, 5,939,291 and 5,942,391, GB ApplicationNo. 2 202 328, and in PCT Application No. PCT/US89/01025, each of whichis incorporated herein by reference in its entirety.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, mayalso be used as an amplification method in the present invention. Inthis method, a replicative sequence of RNA that has a regioncomplementary to that of a target is added to a sample in the presenceof an RNA polymerase. The polymerase will copy the replicative sequencewhich may then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention (Walker et al., 1992). StrandDisplacement Amplification (SDA), disclosed in U.S. Pat. No. 5,916,779,is another method of carrying out isothermal amplification of nucleicacids which involves multiple rounds of strand displacement andsynthesis, i.e., nick translation.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (Kwoh et al., 1989; Gingeras et al., PCTApplication WO 88/10315, incorporated herein by reference in theirentirety). European Application No. 329 822 disclose a nucleic acidamplification process involving cyclically synthesizing single-strandedRNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA), which may be usedin accordance with the present invention.

PCT Application WO 89/06700 (incorporated herein by reference in itsentirety) disclose a nucleic acid sequence amplification scheme based onthe hybridization of a promoter region/primer sequence to a targetsingle-stranded DNA (“ssDNA”) followed by transcription of many RNAcopies of the sequence. This scheme is not cyclic, i.e., new templatesare not produced from the resultant RNA transcripts. Other amplificationmethods include “race” and “one-sided PCR” (Frohman, 1990; Ohara et al.,1989).

C. Detection of Nucleic Acids

Following any amplification for TFAP2A, E2F5, or CA125, it may bedesirable to separate the amplification product from the template and/orthe excess primer. In one embodiment, amplification products areseparated by agarose, agarose-acrylamide or polyacrylamide gelelectrophoresis using standard methods (Sambrook et al., 1989).Separated amplification products may be cut out and eluted from the gelfor further manipulation. Using low melting point agarose gels, theseparated band may be removed by heating the gel, followed by extractionof the nucleic acid.

Separation of nucleic acids may also be effected by chromatographictechniques known in art. There are many kinds of chromatography whichmay be used in the practice of the present invention, includingadsorption, partition, ion-exchange, hydroxylapatite, molecular sieve,reverse-phase, column, paper, thin-layer, and gas chromatography as wellas HPLC.

In certain embodiments, the amplification products are visualized. Atypical visualization method involves staining of a gel with ethidiumbromide and visualization of bands under UV light. Alternatively, if theamplification products are integrally labeled with radio- orfluorometrically-labeled nucleotides, the separated amplificationproducts can be exposed to x-ray film or visualized under theappropriate excitatory spectra.

In one embodiment, following separation of amplification products, alabeled nucleic acid probe is brought into contact with the amplifiedmarker sequence. The probe preferably is conjugated to a chromophore butmay be radiolabeled. In another embodiment, the probe is conjugated to abinding partner, such as an antibody or biotin, or another bindingpartner carrying a detectable moiety.

In particular embodiments, detection is by Southern blotting andhybridization with a labeled probe. The techniques involved in Southernblotting are well known to those of skill in the art (see Sambrook etal., 1989). One example of the foregoing is described in U.S. Pat. No.5,279,721, incorporated by reference herein, which discloses anapparatus and method for the automated electrophoresis and transfer ofnucleic acids. The apparatus permits electrophoresis and blottingwithout external manipulation of the gel and is ideally suited tocarrying out methods according to the present invention.

Other methods of nucleic acid detection that may be used in the practiceof the instant invention are disclosed in U.S. Pat. Nos. 5,840,873,5,843,640, 5,843,651, 5,846,708, 5,846,717, 5,846,726, 5,846,729,5,849,487, 5,853,990, 5,853,992, 5,853,993, 5,856,092, 5,861,244,5,863,732, 5,863,753, 5,866,331, 5,905,024, 5,910,407, 5,912,124,5,912,145, 5,919,630, 5,925,517, 5,928,862, 5,928,869, 5,929,227,5,932,413 and 5,935,791, each of which is incorporated herein byreference.

VI. Methods of Measuring Protein Expression

In certain embodiments, immunodetection methods are used to measureexpression levels, such as increased expression levels of TFAP2A, E2F5,and CA125. Examples of particular immunodetection methods include enzymelinked immunosorbent assay (ELISA), radioimmunoassay (RIA),immunoradiometric assay, immunohistochemistry, fluoroimmunoassay,chemiluminescent assay, bioluminescent assay, Western blot, and soforth. The steps of various useful immunodetection methods have beendescribed in the scientific literature, such as, e.g., Doolittle andBen-Zeev, 1999; Gulbis and Galand, 1993; De Jager et al., 1993; Nakamuraet al., 1987, each incorporated herein by reference.

In general, the immunobinding methods involve measurement of theformation of immunocomplexes. Other methods include methods forisolating and purifying the TFAP2A, E2F5, and CA125 protein from a cell,tissue or organism's samples (such as blood, for example). In theseinstances, the antibody removes the antigenic TFAP2A, E2F5, and CA125protein or message from a sample. The antibody will preferably be linkedto a solid support, such as in the form of a column matrix, and thesample suspected of containing the message, protein, polypeptide and/orpeptide antigenic component will be applied to the immobilized antibody.The unwanted components will be washed from the column, leaving theantigen immunocomplexed to the immobilized antibody to be eluted.

The immunobinding methods also include methods for detecting andquantifying the amount of an antigen component in a sample and thedetection and quantification of any immune complexes formed during thebinding process. Here, one would obtain a sample suspected of containingan antigen, and contact the sample with an antibody against the TFAP2A,E2F5, and CA125 protein, and then detect and quantify the amount ofimmune complexes formed under the specific conditions.

In terms of antigen detection, the biological sample analyzed may be anysample that is suspected of containing an antigen, such as, for example,a tissue section or specimen, a homogenized tissue extract, a cell, anorganelle, separated and/or purified forms of any of the aboveantigen-containing compositions, or even any biological fluid that comesinto contact with the cell or tissue, including blood and/or serum,although tissue samples or extracts are preferred.

Contacting the chosen biological sample with the antibody undereffective conditions and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) is generally amatter of simply adding the antibody composition to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to, any TFAP2A,E2F5, and CA125 antigens present. After this time, the sample-antibodycomposition, such as a tissue section, ELISA plate, dot blot or westernblot, will generally be washed to remove any non-specifically boundantibody species, allowing only those antibodies specifically boundwithin the primary immune complexes to be detected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. U.S. Patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. Of course, one may find additional advantages through theuse of a secondary binding ligand such as a second antibody and/or abiotin/avidin ligand binding arrangement, as is known in the art.

Any TFAP2A, E2F5, and CA125 antibody employed in the detection mayitself be linked to a detectable label, wherein one would then simplydetect this label, thereby allowing the amount of the primary immunecomplexes in the composition to be determined. Alternatively, the firstantibody that becomes bound within the primary immune complexes may bedetected by means of a second binding ligand that has binding affinityfor the antibody. In these cases, the second binding ligand may belinked to a detectable label. The second binding ligand is itself oftenan antibody, which may thus be termed a “secondary” antibody. Theprimary immune complexes are contacted with the labeled, secondarybinding ligand, or antibody, under effective conditions and for a periodof time sufficient to allow the formation of secondary immune complexes.The secondary immune complexes are then generally washed to remove anynon-specifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the antibody is used to form secondary immunecomplexes, as described above. After washing, the secondary immunecomplexes are contacted with a third binding ligand or antibody that hasbinding affinity for the second antibody, again under effectiveconditions and for a period of time sufficient to allow the formation ofimmune complexes (tertiary immune complexes). The third ligand orantibody is linked to a detectable label, allowing detection of thetertiary immune complexes thus formed. This system may provide forsignal amplification if this is desired.

One method of immunodetection designed by Charles Cantor uses twodifferent antibodies. A first step biotinylated, monoclonal orpolyclonal antibody is used to detect the target antigen(s), and asecond step antibody is then used to detect the biotin attached to thecomplexed biotin. In that method the sample to be tested is firstincubated in a solution containing the first step antibody. If thetarget antigen is present, some of the antibody binds to the antigen toform a biotinylated antibody/antigen complex. The antibody/antigencomplex is then amplified by incubation in successive solutions ofstreptavidin (or avidin), biotinylated DNA, and/or complementarybiotinylated DNA, with each step adding additional biotin sites to theantibody/antigen complex. The amplification steps are repeated until asuitable level of amplification is achieved, at which point the sampleis incubated in a solution containing the second step antibody againstbiotin. This second step antibody is labeled, as for example with anenzyme that can be used to detect the presence of the antibody/antigencomplex by histoenzymology using a chromogen substrate. With suitableamplification, a conjugate can be produced which is macroscopicallyvisible.

Another known method of immunodetection takes advantage of theimmuno-PCR (Polymerase Chain Reaction) methodology. The PCR method issimilar to the Cantor method up to the incubation with biotinylated DNA,however, instead of using multiple rounds of streptavidin andbiotinylated DNA incubation, the DNA/biotin/streptavidin/antibodycomplex is washed out with a low pH or high salt buffer that releasesthe antibody. The resulting wash solution is then used to carry out aPCR reaction with suitable primers with appropriate controls. At leastin theory, the enormous amplification capability and specificity of PCRcan be utilized to detect a single antigen molecule.

A. Western Blot Analysis

Western blot analysis is an established technique that is commonlyemployed for analyzing and identifying proteins. The proteins are firstseparated by electrophoresis in polyacrylamide gel, then transferred(“blotted”) onto a nitrocellulose membrane or treated paper, where theybind in the same pattern as they formed in the gel. The antigen isoverlaid first with antibody, then with anti-immunoglobulin or protein Alabeled with a radioisotope, fluorescent dye, or enzyme. One of ordinaryskill in the art would be familiar with this commonly used technique forquantifying protein in a sample.

B. ELISAs

As detailed above, immunoassays, in their most simple and/or directsense, are binding assays. Certain preferred immunoassays are thevarious types of enzyme linked immunosorbent assays (ELISAs) and/orradioimmunoassays (RIA) known in the art. Immunohistochemical detectionusing tissue sections is also particularly useful. However, it will bereadily appreciated that detection is not limited to such techniques,and/or western blotting, dot blotting, FACS analyses, and/or the likemay also be used. One of ordinary skill in the art would be familiarwith use of ELISAs and other immunohistochemical assays.

C. Immunohistochemistry

The antibodies of the present invention may also be used in conjunctionwith both fresh-frozen and/or formalin-fixed, paraffin-embedded tissueblocks, such as blocks prepared from a tumor biopsy, prepared for studyby immunohistochemistry (IHC). The method of preparing tissue blocksfrom these particulate specimens has been successfully used in previousIHC studies of various prognostic factors, and/or is well known to thoseof skill in the art (Brown et al., 1990; Abbondanzo et al., 1999; Allredet al., 1990).

Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen“pulverized” tissue at room temperature in phosphate buffered saline(PBS) in small plastic capsules; pelleting the particles bycentrifugation; resuspending them in a viscous embedding medium (OCT);inverting the capsule and/or pelleting again by centrifugation;snap-freezing in 70° C. isopentane; cutting the plastic capsule and/orremoving the frozen cylinder of tissue; securing the tissue cylinder ona cryostat microtome chuck; and/or cutting 25-50 serial sections.

Permanent-sections may be prepared by a similar method involvingrehydration of the 50 mg sample in a plastic microfuge tube; pelleting;resuspending in 10% formalin for 4 hours fixation; washing/pelleting;resuspending in warm 2.5% agar; pelleting; cooling in ice water toharden the agar; removing the tissue/agar block from the tube;infiltrating and/or embedding the block in paraffin; and/or cutting upto 50 serial permanent sections.

D. Protein Array Technology

Protein array technology allows high-throughput screening for geneexpression and molecular interactions. Protein arrays appear as new andversatile tools in functional genomics, enabling the translation of geneexpression patterns of normal and diseased tissues into protein productcatalog. Protein function, such as enzyme activity, antibodyspecificity, and other ligand-receptor interactions and binding ofnucleic acids or small molecules can be analyzed on a whole-genomelevel.

1. Protein Biochip Assays

These arrays, which contain thousands of different proteins orantibodies spotted onto glass slides or immobilized in tiny wells, allowone to examine the biochemical activities and binding profiles of alarge number of proteins at once. To examine protein interactions withsuch an array, a labeled protein is incubated with each of the targetproteins immobilized on the slide, and then one determines which of themany proteins the labeled molecule binds.

The basic construction of protein chips has some similarities to DNAchips, such as the use of a glass or plastic surface dotted with anarray of molecules. These molecules can be DNA or antibodies that aredesigned to capture proteins. Defined quantities of proteins areimmobilized on each spot, while retaining some activity of the protein.With fluorescent markers or other methods of detection revealing thespots that have captured these proteins, protein microarrays are beingused as powerful tools in high-throughput proteomics and drug discovery.

Glass slides are still widely used, since they are inexpensive andcompatible with standard microarrayer and detection equipment. However,their limitations include multiple-based reactions, high evaporationrates, and possible cross-contamination.

Matrix slides offer a number of advantages, such as reduced evaporationand no possibility of cross-contamination, but they are expensive.Nanochips for proteomics have the same advantages, in addition toreduced cost and the capability of multiple-component reactions.

The earliest and best-known protein chip is the ProteinChip by CiphergenBiosystems Inc. (Fremont, Calif.). The ProteinChip is based on thesurface-enhanced laser desorption and ionization (SELDI) process. Knownproteins are analyzed using functional assays that are on the chip. Forexample, chip surfaces can contain enzymes, receptor proteins, orantibodies that enable researchers to conduct protein-proteininteraction studies, ligand binding studies, or immunoassays. Withstate-of-the-art ion optic and laser optic technologies, the ProteinChipsystem detects proteins ranging from small peptides of less than 1000 Daup to proteins of 300 kDa and calculates the mass based ontime-of-flight (TOF).

The ProteinChip biomarker system is the first protein biochip-basedsystem that enables biomarker pattern recognition analysis to be done.This system allows researchers to address important clinical questionsby investigating the proteome from a range of crude clinical samples(i.e., laser capture microdissected cells, biopsies, tissue, urine, andserum). The system also utilizes biomarker pattern software thatautomates pattern recognition-based statistical analysis methods tocorrelate protein expression patterns from clinical samples with diseasephenotypes.

Some systems can perform biomarker discovery in days and validation oflarge sample sets within weeks. Its robotics system accessory automatessample processing, allowing hundreds of samples to be run per week andenabling a sufficient number of samples to be run, which provides highstatistical confidence in comprehensive studies for marker discovery andvalidation.

2. Microfluidic Chip-Based Immunoassays

Microfluidics is one of the most important innovations in biochiptechnology. Since microfluidic chips can be combined with massspectrometric analysis, a microfluidic device has been devised in whichan electrospray interface to a mass spectrometer is integrated with acapillary electrophoresis channel, an injector, and a protein digestionbed on a monolithic substrate (Wang et al., 2000). This chip thusprovides a convenient platform for automated sample processing inproteomics applications.

These chips can also analyze expression levels of serum proteins withdetection limits comparable to commercial enzyme-linked immunosorbentassays, with the advantage that the required volume sample is markedlylower compared with conventional technologies.

Biosite (San Diego) manufactures the Triage protein chip thatsimultaneously measures 100 different proteins by immunoassays. TheTriage protein chip immunoassays are performed in a microfluidic plasticchip, and the results are achieved in 15 minutes with picomolarsensitivities. Microfluidic fluid flow is controlled in the protein chipby the surface architecture and surface hydrophobicity in themicrocapillaries. The immunoassays utilize high-affinity antibodies anda near-infrared fluorescent label, which is read by a fluorometer.

3. Tissue Microarray Technology

Tissue microarray technology provides a high-throughput approach forlinking genes and gene products with normal and disease tissues at thecellular level in a parallel fashion. Compared with classical in situtechnologies in molecular pathology that are very time-consuming, tissuemicroarrays provide increased throughput in two ways: up to 1000 tissuespecimens can be analyzed in a single experiment, either at the DNA,RNA, or protein level; and tens of thousands of replicate tissuemicroarrays can be generated from a set of tissues. This processprovides a template for analyzing many more biomarkers than has everbeen possible previously in a clinical setting, even using archival,formalin-fixed specimens.

4. Nanoscale Protein Analysis

Most current protocols including protein purification and automatedidentification schemes yield low recoveries that limit the overallprocess in terms of sensitivity and speed. Such low protein yields andproteins that can only be isolated from limited source material (e.g.,biopsies) can be subjected to nanoscale protein analysis: a nanocaptureof specific proteins and complexes, and optimization of all subsequentsample-handling steps, leading to a mass analysis of peptide fragments.This focused approach, also termed targeted proteomics, involvesexamining subsets of the proteome (e.g., those proteins that arespecifically modified, bind to a particular DNA sequence, or exist asmembers of higher-order complexes or any combination thereof). Thisapproach is used to identify genetic determinants of cancer that altercellular physiology and respond to agonists.

A new detection technique called multiphoton detection, by Biotrace Inc.(Cincinnati), can quantify subzeptomole amounts of proteins and will beused for diagnostic proteomics, particularly for cytokines and otherlow-abundance proteins. Biotrace is also developing supersensitiveprotein biochips to detect concentrations of proteins as low as 5 fg/ml(0.2 attomole/ml), thereby permitting sensitivity that is 1000 timesgreater than current protein biochips.

VII. Kits of the Invention

All of the essential materials and/or reagents required for detectingTFA2P2A, E2F5, and/or CA125 in a sample may be assembled together in akit. In particular embodiments, the kit comprises respective antibodiesto TFA2P2A, E2F5, and/or CA125. This may comprise a probe or primersdesigned to hybridize specifically to individual nucleic acids ofinterest in the practice of the present invention, including TFA2P2A,E2F5, and/or CA125, in some cases. Also included may be enzymes suitablefor amplifying nucleic acids, including various polymerases (reversetranscriptase, Taq, etc.), deoxynucleotides and buffers to provide thenecessary reaction mixture for amplification. Such kits may also includeenzymes and other reagents suitable for detection of specific nucleicacids or amplification products. Such kits generally will comprise, insuitable means, distinct containers for each individual reagent orenzyme as well as for each probe or primer pair.

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, a composition of the invention may be comprised ina kit. The components of the kits may be packaged either in aqueousmedia or in lyophilized form. The container means of the kits willgenerally include at least one vial, test tube, flask, bottle, syringeor other container means, into which a component may be placed, andpreferably, suitably aliquoted. Where there is more than one componentin the kit, the kit also will generally contain a second, third or otheradditional container into which the additional components may beseparately placed. However, various combinations of components may becomprised in a vial. The kits of the present invention also willtypically include a means for containing the composition and any otherreagent containers in close confinement for commercial sale. Suchcontainers may include injection or blow molded plastic containers intowhich the desired vials are retained, for example.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. In specific cases, thecontainer means may itself be a syringe, pipette, and/or other such likeapparatus, from which the formulation may be applied to the pancreas,for example. However, the components of the kit may be provided as driedpowder(s). When reagents and/or components are provided as a dry powder,the powder can be reconstituted by the addition of a suitable solvent.It is envisioned that the solvent may also be provided in anothercontainer means.

VIII. TFAP2A as a Marker for Other Cancers

In some embodiments of the invention, TFAP2A level is assayed fordetection of a cancer other than ovarian cancer. Although TFAP2A may beindicative of having or being at greater risk for having any cancerother than ovarian cancer, in specific embodiments, the cancer is of theuterus, breast, lung, prostate, colon, brain, bone, liver, pancreas,cervix, testes, spleen, skin, gall bladder, esophagus, bladder, kidney,thyroid, blood, and so forth. In specific embodiments, level of TFAP2Aprotein and/or mRNA is increased or decreased in a cancer other thanovarian cancer when compared to a control. In specific embodiments,markers in addition to TFAP2A are employed with TFAP2A to detect thepresence or increased risk of having a particular type of cancer.Exemplary other tumor markers include prostate-specific antigen (PSA)for prostate cancer, calcitonin for medullary thyroid cancer,alpha-fetoprotein (AFP) for liver cancer and human chorionicgonadotropin (HCG) for germ cell tumors, such as testicular cancer andovarian cancer.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Molecular Biomarker Set for Detection of Ovarian Cancer

The inventors compared the expression levels of genes in canceroustissues categorized as stage IA, IC, and IIIC. FIG. 1 demonstrates thatthe expression of TFAP2A gene is higher in early stages (stages IA andIC) as well as advanced stage (IIIC) of the ovarian cancer as comparedto the normal tissue (FIG. 1).

Kothandaraman et al. (2010) described E2F5 as a biomarker of ovariancancer and they showed that the use of E2F5 in combination with CA125increased sensitivity of ovarian cancer detection to 97.9% (an increasefrom 87.5% if only CA125 is used) in the case when the status of any ofE2F5 or CA125 is confirmed. If the status of both E2F5 and CA125 isconfirmed, the specificity of ovarian cancer detection increases to72.5% (an increase from 55% if only CA125 is used) in a subset ofpatients. This study is the evidence that the combination markers can bemore useful in diagnosing ovarian cancer. To further enhance theaccuracy of ovarian cancer detection at early stages, the inventions setforth that the combination of TFAP2A, E2F5, and CA125 are useful forincreased accuracy in detection of ovarian cancer. Two of thesebiomarkers i.e. E2F5 and CA125 have already been published and thecomputational analysis has detected TFAP2A as a biomarker of ovariancancer.

The inventors describe here the identification of a new biomarker usingcomputational methods that is useful for diagnosis of ovarian cancer.The results are based on a set A of 323 experimentally validated OCimplicated genes compiled from literature. For this gene set, theinventors determined putative transcription factors (TFs) that controlgene activation. The inventors ranked these TFs based on the number ofgenes they control in set A. In this way, the inventors selectedtop-ranked TFs as potential biomarkers for set A. Comparing theexpression of top-ranked TFs in OC and normal cases based on publisheddata (Lu et al., 2004, Hendrix et al., 2006, Adib et al., 2004), weidentified TF named TFAP2A as a new biomarker for detection of ovariancancer. The newly identified biomarker TFAP2A was compared with knownmarker CA125 in terms of expression in cancer patients in relevantpublished datasets.

Example 2 Exemplary Clinical Application of the Invention

In certain embodiments of the invention, an individual that is suspectedof having ovarian cancer (for example, because of one or more testresults and/or symptoms of ovarian cancer; an individual that is at riskfor developing ovarian cancer (for example, because of a familyhistory); an individual presently or previously on therapy for ovariancancer; or an individual practicing routine health checks for ovariancancer is subjected to methods of the invention for determiningexpression levels of TFAP2A, E2F5, and/or CA125.

The individual provides a sample to be tested. The sample may beprocessed by the same person or organization that obtains the sample, orthe sample may be forwarded to another party for assaying or may bestored appropriately. The sample may be a biological fluid sample suchas a blood sample, although in some cases the sample is an ovarianbiopsy. The sample may be further processed, for example, blood may befractionated, or ovarian tissue may have nucleic acid extracted. Therespective mRNA or protein may be obtained from the sample by routinemethods in the art, and is level is obtained and compared to a control,which may also be referred to as a standard. The control may compriselevels of TFAP2A, E2F5, and CA125 that are obtained from one or morenormal individuals (persons without the respective cancer), and theselevels may be obtained from the respective fluid or tissue in the normalindividual(s). In the case of ovarian biopsy, for example, the level maybe obtained from ovarian tissue in a region of the ovary being testedthat detectably lacks cancerous cells and/or may be from the secondovary of the individual than the one being tested. In other ovarianbiopsy cases, the level is obtained from one or more normal individuals.

Example 3 Combinations of Biomarkers for Detection of Ovarian Cancer

The present example provides exemplary methods demonstrating detectionof at least one ovarian cancer markers.

Samples

The RNA samples for 100 individuals were obtained from a commercialsupplier and the distribution of samples is as follows:

TABLE 1 Distribution of tissue samples used for gene expression studySample detail Sample Numbers Ovarian Cancer (OC) 60 Breast cancer 10Cervix cancer 10 Uterus cancer 10 Non-cancerous ovary 10

The RNA was extracted from the respective tissues by standard means inthe art.

Exemplary Experimental Procedures

The 100 RNA samples underwent quality control (QC) using a bioanalyzerprior to cDNA synthesis with the ABI high capacity cDNA synthesis kit.The cDNA was then analyzed against five commercially available TaqMan®gene expression assays (Table 2) with the ABI TaqMan0 gene expressionprotocol using the 7900HT RT-PCR instrument.

TABLE 2 TaqMan ® Assays used in gene expression study Inventory CodeGene Name Gene Classification 1 Hs01029413_m1 TFAP2A TranscriptionFactor AP-2 α 2 Hs00231092_m1 E2F5 E2F Transcription Factor 5, p130binding 3 Hs03928990_g1 RN18S1 RNA, 18S ribosomal 1 (exemplaryhousekeeping gene) 4 Hs03929097_g1 GAPDH Glyceraldehyde-3-phosphatedehydrogenase (exemplary housekeeping gene) 5 Hs01065189_m1 MUC16(CA125) Mucin 16, cell surface associated

cDNA Synthesis and QC

cDNA synthesis was performed according to the manufacturer'sinstructions using the ABI High Capacity cDNA synthesis kits and QC wascarried out using the Nanodrop instrument. Good quality cDNA wasproduced with an average cDNA yield of ˜1490 ng/μl per sample. The260/280 and 260/230 ratios were within the optimal range with averagevalues of 1.8 and 2.0, respectively.

qRT-PCR QC and Analysis

A 1 μl aliquot from each of the 100 samples was pooled to create astandard sample. The standard sample was further subjected to QC withthe nanodrop and diluted to make up a dilution series for standard curveanalysis. A standard curve was generated for each gene and the TaqMan®assays were all found to be within 100±10% efficiency (Table 3), where aslope of −3.32 indicates an assay with 100% efficiency.

TABLE 3 Standard Curve efficiencies for the five genes underinvestigation Detector Name Slope R² Hs01029413_m1_TFAPZA −3.60171250.998070 Hs00231092_m1_E2F5 −3.5165527 0.9947431 Hs03928990_g1_RN1851−3.56650 0.9986217 Hs03929097_g1_GAPDH −3.3637228 0.9978328Hs01065189_m1 MUC16 −3.5434833 0.99704236 (CA125)

For the experimental procedure, each sample was assayed in triplicate tolimit technical variation. The assay comprised of cDNA samples mixedtogether with a cocktail containing the primer and probe pairs of thespecific gene to be investigated. Samples were loaded onto a 384-wellopti-clear PCR plate and the assay was performed under the ABIprescribed conditions on the 7900HT RT-PCR. Primary data analysis wasexecuted with the use of the ABI SDS v2.3 software package.

Results

Average Ct values for each gene were provided for further analysis. Ctrefers to cycle threshold and is defined as the number of cyclesrequired for a fluorescent signal to cross a threshold. The threshold isthe point where signal exceeds background level. The amount of amplifiednucleic acid in the sample is inversely proportional to Ct (i.e. thelower the Ct level the greater the amount of target nucleic acid in thesample).

The inventors used ΔΔCt method to calculate the expression value foreach gene. The method is summarized as follows:

Step 1:ΔCt _((Target gene)) =Ct _((Target gene)) −Ct _((housekeeping gene))

Step 2:ΔΔCt _((OC)) =ΔCt _((Target gene in OC)) −ΔCt_((Target gene in non-OC (average)))ΔΔCt _((non-OC)) =ΔCt _((Target gene in non-OC)) −ΔCt_((Target gene in non-OC (average)))

Determining an accurate combination of biomarkers:

The expression data of target genes (TFAP2A, E2F5 and CA125) normalizedagainst RN18S1 reference gene (control) (according to Steps 1 and 2above) was used for predicting the accuracy of the diagnosis of OC usingbiomarkers in different combinations. Table 4 represents the results ofthis analysis. The terms “se”, “sp” and “acc” denote sensitivity,specificity and accuracy, respectively. These parameters are calculatedas follows:se=tp/(tp+fn),sp=tn/(tn+fp),acc=(tp+tn)/(tp+fn+tn+fp),

where tp, tn, fp, fn stand for true positive, true negative, falsepositive and false negative outcome of diagnoses.

tp means that the OC case is diagnosed as being OC;

tn means that the non-OC case is diagnosed as being non-OC;

fp means that the non-OC case is diagnosed falsely as being OC;

fn means that the OC case is falsely diagnosed as being non-OC.

The columns in Table 4 represent the following:

‘max se−(1−sp)’ is a measure that shows how well OC cases are diagnosedrelative to non-OC cases. (1−sp) shows the proportion of the non-OCcases diagnosed wrongly as being OC. Thus the difference between se and(1−sp) is a good indicator of the quality of diagnosis. The higher thevalue of ‘se−(1−sp)’, the more useful are the biomarkers.

Threshold for TFAP2A: the threshold th. In specific embodiments,ΔΔCt_((TFAP2A))>th in different combinations of biomarkers is useful fordiagnosis of OC;

Threshold for E2F5: the threshold th. In specific embodiments,ΔΔCt_((E2F5))>th in different combinations of biomarkers is useful fordiagnosis of OC;

Threshold for CA125: the threshold th. In specific embodiments,ΔΔCt_((CA125))>th in different combinations of biomarkers is useful fordiagnosis of OC;

For different combinations of biomarkers (see Table 4), the thresholdvalues were obtained by searching for the combination of thresholds thatwill maximize ‘se−(1−sp)’ subject to condition that this threshold hasvalue between −10 and 10. The inventors used Direct Search optimizationmethod from the Global Optimization Toolbox of Matlab package release2011b (commercially available from Mathworks, Natick, Mass., USA).

N/A: means that particular biomarker is not used in the test.

TABLE 4 The diagnostic value of different combinations of biomarkers wascalculated based on threshold of ΔΔCt of each target biomarker. ‘&’means logical ‘AND’ max threshold threshold threshold ‘|’ means logicalse-(1- for for for ‘OR’ sp) Sensitivity Specificity AccuracyΔΔCt_((TFAP2A)) ΔΔCt_((E2F5)) ΔΔCt_((CA125)) TFAP2A & E2F5 & 0.4333330.683333 0.75 0.71 −1.59 −1.16 −2.86 CA125 TFAP2A | E2F5 & 0.3833330.883333 0.5 0.73 7.13 −0.85 −2.66 CA125 TFAP2A & E2F5 | 0.375 0.650.725 0.68 −0.46 −0.58 3.51 CA125 TFAP2A & (E2F5 | 0.341667 0.7166670.625 0.68 −1.58 9.027 −2.87 CA125) TFAP2A | E2F5 | 0.3 0.8 0.5 0.68−0.38 0.61 3.6 CA125 E2F5 & CA125 0.383333 0.883333 0.5 0.73 N/A −0.85−2.76 TFAP2A & CA125 0.341667 0.716667 0.625 0.68 −1.62 N/A −2.87 TFAP2A& E2F5 0.308333 0.633333 0.675 0.65 −1.933 −0.526 N/A E2F5 | CA1250.291667 0.866667 0.425 0.69 N/A −0.526 1.95 TFAP2A | E2F5 0.2666670.816667 0.45 0.67 8.62 −0.524 N/A TFAP2A | CA125 0.258333 0.7833330.475 0.66 −0.75 N/A 3.53 E2F5 0.266667 0.816667 0.45 0.67 N/A −0.526N/A CA125 0.241667 0.916667 0.325 0.68 N A N/A −2.474 TFAP2A 0.1833330.733333 0.45 0.62 −1.59 N/A N/A

Table 4 shows that combination of TFAP2A & E2F5 & CA125 has max value ofse−(1−sp)=0.433 (first data row in Table 4) for the samples analyzed,which is highest than any other combination of biomarkers. This showsthat the combination of these three biomarkers provides most confidencein diagnosing correctly 68% of OC cases and identifies correctly 75% ofnon-ovarian cases. Individually, each biomarker (last three rows ofTable 4) E2F5, CA125 and TFAP2A can only identify 45%, 32% and 45% ofnon-OC cases, respectively, since the specificity of individualbiomarker is very low. For example, CA125, which is a routinely usedbiomarker, could only classify 32% of cases as non-OC cases, whereascombination of the three exemplary biomarkers could identify 75% ofnon-OC cases. Therefore, the proposed combination of biomarkers (TFAP2A,CA125 and E2F5) has increased the overall accuracy to detect OC cases aswell as non-OC cases as compared to other combinations ofbiomarkers/individual biomarkers. However, in alternative embodiments,TFAP2A alone is accurate to identify the presence of OC or risk ofdeveloping OC, or other cancers.

Example 4 Analysis Summary of Expression Behavior of Biomarkers inBreast, Cervix and Uterus Cancers

This example describes the expression behavior of three biomarkers(TFAP2A, E2F5 and CA125) in samples obtained from breast, cervix anduterine cancers. The sample size for each cancer type and normal is 10.The expression level for each biomarker was assayed and quantitatedessentially as described in Example 3. The following graphs representthe expression values observed for normal and cancers calculated asfollows:

Step 1:ΔCt _((Target gene)) −Ct _((Target gene)) −Ct _((housekeeping gene))

Step 2:ΔΔCt _((cancer)) =ΔCt _((Target gene in cancer)) −ΔCt_((Target gene in normal (average)))ΔΔCt _((normal)) =ΔCt _((Target gene in normal)) −ΔCt_((Target gene in normal (average)))

The relative expression of each biomarker shown in the figures iscalculated as 2^((−ΔΔct)) (Arocho et al., 2006)

Exemplary Results

The results of the analysis are represented in the form of bar graphsfor each cancer type and normal samples.

FIG. 2 shows a bar graph representing the expression pattern of threebiomarkers in normal samples. The Y-axis represents the expressionlevels of biomarkers in each normal sample. The X-axis representsindividual samples. The expression of TFAP2A was very low orundetectable in 8/10 samples, the expression of E2F5 was low in most ofthe normal samples, whereas the expression of CA125 was low or was notdetected in 8/10 samples.

FIG. 3 shows the expression pattern of the three biomarkers in breastcancer samples. The Y-axis represents the expression levels ofbiomarkers in each breast cancer sample. The X-axis representsindividual samples. In breast cancer samples, the expression level ofCA125 was higher than the control in 7/10 samples. Zero on the Y-axis isthe lowest boundary on expression, i.e. it means no expression.

FIG. 4 shows the expression pattern of the three biomarkers in cervixcancer samples. The Y-axis represents the expression levels ofbiomarkers in each cervix cancer sample. The X-axis representsindividual samples. Six out of 10 cervix cancer samples had higherexpression levels than the control for all three biomarkers.

FIG. 5 demonstrates the expression pattern of the three biomarkers inuterus cancer samples. The Y-axis represents the expression levels ofbiomarkers in each uterus cancer sample. The X-axis representsindividual samples.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

What is claimed is:
 1. A method of identifying an individual to betreated for ovarian cancer, consisting of: a) detecting expressionlevels of TFAP2A, E2F5, and CA125from a sample obtained from theindividual; b) selecting the individual for treatment when expressionlevels of TFAP2A, E2F5, and CA125 are higher relative to the expressionlevels of TFAP2A, E2F5, and CA125 from a control; and c) administeringto the individual a treatment for ovarian cancer, wherein the treatmentfor ovarian cancer is selected from the group consisting of surgery,radiation, and chemotherapy.
 2. The method of claim 1, wherein theexpression levels are determined by protein level or mRNA level ofTFAP2A, CA125 and E2F5.
 3. The method of claim 2, wherein the proteinlevel of TFAP2A, CA125 and E2F5 is determined with an antibody.
 4. Themethod of claim 2, wherein the mRNA levels of TFAP2A, CA125 and E2F5from the individual are determined.
 5. The method of claim 2, whereinthe mRNA level is determined from ovarian tissue or blood from theindividual.
 6. The method of claim 1, wherein the control is tissue orblood from one or more normal individuals or is from tissue or bloodfrom the individual.
 7. The method of claim 1, wherein the methoddetects the stage of ovarian cancer in the individual.
 8. The method ofclaim 7, wherein the stage is stage IA, IC, IIIC, or a combinationthereof.
 9. A method of identifying an individual to be treated forovarian cancer, consisting of: a) detecting expression levels of TFAP2A,E2F5, and CA125 from a sample obtained from the individual; b) selectingthe individual for treatment when expression levels of TFAP2A, E2F5, andCA125 are higher relative to the expression levels of TFAP2A, E2F5, andCA125 from a control; c) performing an additional ovarian cancerdetection method; and d) administering to the individual a treatment forovarian cancer, wherein the treatment for ovarian cancer is selectedfrom the group consisting of surgery, radiation, and chemotherapy, andwherein the additional ovarian cancer detection method is selected fromthe group consisting of palpitation, ultrasound, magnetic resonanceimaging, X-ray, CT scan, blood testing, and biopsy.
 10. The method ofclaim 1, wherein the sample is ovarian tissue, blood, serum, or plasma.11. The method of claim 1, wherein the chemotherapy includesadministering a chemotherapeutic agent selected from the groupconsisting of drugs that contain platinum and taxane compounds.
 12. Themethod of claim 11, wherein the chemotherapeutic agent is selected fromthe group consisting of cisplatin, carboplatin, and paclitaxel.
 13. Themethod of claim 1, wherein chemotherapy comprises administering aneffective amount of melphalan, doxorubicin, altretamine, 5-fluorouracil,topotecan, ifosamide, and etoposide.